Towards Prediction of Metabolic Products of Polyketide Synthases: An In Silico Analysis

Sequence data arising from an increasing number of partial and complete genome projects is revealing the presence of the polyketide synthase (PKS) family of genes not only in microbes and fungi but also in plants and other eukaryotes. PKSs are huge multifunctional megasynthases that use a variety of biosynthetic paradigms to generate enormously diverse arrays of polyketide products that posses several pharmaceutically important properties. The remarkable conservation of these gene clusters across organisms offers abundant scope for obtaining novel insights into PKS biosynthetic code by computational analysis. We have carried out a comprehensive in silico analysis of modular and iterative gene clusters to test whether chemical structures of the secondary metabolites can be predicted from PKS protein sequences. Here, we report the success of our method and demonstrate the feasibility of deciphering the putative metabolic products of uncharacterized PKS clusters found in newly sequenced genomes. Profile Hidden Markov Model analysis has revealed distinct sequence features that can distinguish modular PKS proteins from their iterative counterparts. For iterative PKS proteins, structural models of iterative ketosynthase (KS) domains have revealed novel correlations between the size of the polyketide products and volume of the active site pocket. Furthermore, we have identified key residues in the substrate binding pocket that control the number of chain extensions in iterative PKSs. For modular PKS proteins, we describe for the first time an automated method based on crucial intermolecular contacts that can distinguish the correct biosynthetic order of substrate channeling from a large number of non-cognate combinatorial possibilities. Taken together, our in silico analysis provides valuable clues for formulating rules for predicting polyketide products of iterative as well as modular PKS clusters. These results have promising potential for discovery of novel natural products by genome mining and rational design of novel natural products.

[1]  Corinna Lange,et al.  Genomics-driven discovery of PKS-NRPS hybrid metabolites from Aspergillus nidulans. , 2007, Nature chemical biology.

[2]  David C. Jones,et al.  GenTHREADER: an efficient and reliable protein fold recognition method for genomic sequences. , 1999, Journal of molecular biology.

[3]  S. Kroken,et al.  Phylogenomic analysis of type I polyketide synthase genes in pathogenic and saprobic ascomycetes , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[4]  R. Müller,et al.  Myxobacterial natural product assembly lines: fascinating examples of curious biochemistry. , 2007, Natural product reports.

[5]  Gitanjali Yadav,et al.  NRPS-PKS: a knowledge-based resource for analysis of NRPS/PKS megasynthases , 2004, Nucleic Acids Res..

[6]  E. Ron,et al.  The first gene in the biosynthesis of the polyketide antibiotic TA of Myxococcus xanthus codes for a unique PKS module coupled to a peptide synthetase. , 1999, Journal of molecular biology.

[7]  C. Khosla,et al.  Building-block selectivity of polyketide synthases. , 2003, Current opinion in chemical biology.

[8]  C. R. Davis,et al.  Lovastatin biosynthesis in Aspergillus terreus: characterization of blocked mutants, enzyme activities and a multifunctional polyketide synthase gene. , 1999, Chemistry & biology.

[9]  C. Walsh.  The chemical versatility of natural-product assembly lines. , 2008, Accounts of chemical research.

[10]  B. Shen,et al.  Identification and Localization of the Gene Cluster Encoding Biosynthesis of the Antitumor Macrolactam Leinamycin in Streptomyces atroolivaceus S-140 , 2002, Journal of bacteriology.

[11]  Gabrielle A. Reeves,et al.  Structural diversity of domain superfamilies in the CATH database. , 2006, Journal of molecular biology.

[12]  Ben Shen,et al.  Polyketide biosynthesis beyond the type I, II and III polyketide synthase paradigms. , 2003, Current opinion in chemical biology.

[13]  D. Cane,et al.  Intermodular communication in modular polyketide synthases: structural and mutational analysis of linker mediated protein-protein recognition. , 2003, Journal of the American Chemical Society.

[14]  Sylvie Lautru,et al.  Discovery of a new peptide natural product by Streptomyces coelicolor genome mining , 2005, Nature chemical biology.

[15]  D. Cane,et al.  Assessing the balance between protein-protein interactions and enzyme-substrate interactions in the channeling of intermediates between polyketide synthase modules. , 2001, Journal of the American Chemical Society.

[16]  Jörn Piel,et al.  A polyketide synthase-peptide synthetase gene cluster from an uncultured bacterial symbiont of Paederus beetles , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[17]  Minoru Kanehisa,et al.  Comprehensive analysis of distinctive polyketide and nonribosomal peptide structural motifs encoded in microbial genomes. , 2007, Journal of molecular biology.

[18]  Harald Gross,et al.  The genomisotopic approach: a systematic method to isolate products of orphan biosynthetic gene clusters. , 2007, Chemistry & biology.

[19]  E. Dittmann,et al.  Structural organization of microcystin biosynthesis in Microcystis aeruginosa PCC7806: an integrated peptide-polyketide synthetase system. , 2000, Chemistry & biology.

[20]  W. Timberlake,et al.  The developmentally regulated Aspergillus nidulans wA gene encodes a polypeptide homologous to polyketide and fatty acid synthases , 1992, Molecular and General Genetics MGG.

[21]  S. Ripka,et al.  The multifunctional 6-methylsalicylic acid synthase gene of Penicillium patulum. Its gene structure relative to that of other polyketide synthases. , 1990, European journal of biochemistry.

[22]  Ge Yang,et al.  A polyketide synthase is required for fungal virulence and production of the polyketide T-toxin. , 1996, The Plant cell.

[23]  Katalin F Medzihradszky,et al.  An antibiotic factory caught in action , 2004, Nature Structural &Molecular Biology.

[24]  J R Jacobsen,et al.  Tolerance and specificity of polyketide synthases. , 1999, Annual review of biochemistry.

[25]  M. Hauser,et al.  Molecular Cloning and Characterization ofWdPKS1, a Gene Involved in Dihydroxynaphthalene Melanin Biosynthesis and Virulence in Wangiella(Exophiala) dermatitidis , 2001, Infection and Immunity.

[26]  Mohd Zeeshan Ansari,et al.  Dissecting the mechanism and assembly of a complex virulence mycobacterial lipid. , 2005, Molecular cell.

[27]  Ben Shen,et al.  Microbial genomics for the improvement of natural product discovery. , 2006, Current opinion in microbiology.

[28]  G. Challis,et al.  Predictive, structure-based model of amino acid recognition by nonribosomal peptide synthetase adenylation domains. , 2000, Chemistry & biology.

[29]  J. Thorson,et al.  The Calicheamicin Gene Cluster and Its Iterative Type I Enediyne PKS , 2002, Science.

[30]  Jie Liang,et al.  CASTp: Computed Atlas of Surface Topography of proteins , 2003, Nucleic Acids Res..

[31]  K. Gomi,et al.  Cloning of the polyketide synthase gene atX from Aspergillus terreus and its identification as the 6-Methylsalicylic acid synthase gene by heterologous expression , 1996, Molecular and General Genetics MGG.

[32]  Jason Micklefield,et al.  Mining and engineering natural-product biosynthetic pathways. , 2007, Nature chemical biology.

[33]  A. Sali,et al.  Modeller: generation and refinement of homology-based protein structure models. , 2003, Methods in enzymology.

[34]  Frances M. G. Pearl,et al.  Protein folds, functions and evolution. , 1999, Journal of molecular biology.

[35]  R. Doolittle,et al.  A simple method for displaying the hydropathic character of a protein. , 1982, Journal of molecular biology.

[36]  James Staunton,et al.  Analysis of the biosynthetic gene cluster for the polyether antibiotic monensin in Streptomyces cinnamonensis and evidence for the role of monB and monC genes in oxidative cyclization , 2003, Molecular microbiology.

[37]  Christopher M Thomas,et al.  Characterization of the mupirocin biosynthesis gene cluster from Pseudomonas fluorescens NCIMB 10586. , 2003, Chemistry & biology.

[38]  D. Cane,et al.  Selective protein-protein interactions direct channeling of intermediates between polyketide synthase modules. , 2001, Biochemistry.

[39]  Timm Maier,et al.  The Crystal Structure of a Mammalian Fatty Acid Synthase , 2008, Science.

[40]  H. Yoshikawa,et al.  Molecular cloning and characterization of an ML-236B (compactin) biosynthetic gene cluster in Penicillium citrinum , 2002, Molecular Genetics and Genomics.

[41]  D. Grzegorski,et al.  A melanin polyketide synthase (PKS) gene from Nodulisporium sp. that shows homology to the pks1 gene of Colletotrichum lagenarium , 1999, Molecular and General Genetics MGG.

[42]  M. Daffé,et al.  Identification of the Missing trans-Acting Enoyl Reductase Required for Phthiocerol Dimycocerosate and Phenolglycolipid Biosynthesis in Mycobacterium tuberculosis , 2007, Journal of bacteriology.

[43]  Emmanuel Zazopoulos,et al.  Microbial genomics as a guide to drug discovery and structural elucidation: ECO-02301, a novel antifungal agent, as an example. , 2005, Journal of natural products.

[44]  Gitanjali Yadav,et al.  Computational approach for prediction of domain organization and substrate specificity of modular polyketide synthases. , 2003, Journal of molecular biology.

[45]  Makoto M. Watanabe,et al.  Evidence for Recombination in the Microcystin Synthetase (mcy) Genes ofToxic Cyanobacteria Microcystisspp , 2004, Journal of Molecular Evolution.

[46]  K Schulten,et al.  VMD: visual molecular dynamics. , 1996, Journal of molecular graphics.

[47]  G. Jung,et al.  Biosynthesis of the orthosomycin antibiotic avilamycin A: deductions from the molecular analysis of the avi biosynthetic gene cluster of Streptomyces viridochromogenes Tü57 and production of new antibiotics. , 2001, Chemistry & biology.

[48]  Philip E. Bourne,et al.  A database and tools for 3-D protein structure comparison and alignment using the Combinatorial Extension (CE) algorithm , 2001, Nucleic Acids Res..

[49]  Sean R. Eddy,et al.  Profile hidden Markov models , 1998, Bioinform..

[50]  C. Hertweck,et al.  Molecular analysis of the benastatin biosynthetic pathway and genetic engineering of altered fatty acid-polyketide hybrids. , 2007, Journal of the American Chemical Society.

[51]  Gitanjali Yadav,et al.  Novel Intermolecular Iterative Mechanism for Biosynthesis of Mycoketide Catalyzed by a Bimodular Polyketide Synthase , 2008, PLoS biology.

[52]  T. Leonard,et al.  Characterization of the polyketide synthase gene (pksL1) required for aflatoxin biosynthesis in Aspergillus parasiticus , 1995, Journal of bacteriology.

[53]  Patrice Gouet,et al.  ESPript: analysis of multiple sequence alignments in PostScript , 1999, Bioinform..

[54]  Z. Deng,et al.  'Streptomyces nanchangensis', a producer of the insecticidal polyether antibiotic nanchangmycin and the antiparasitic macrolide meilingmycin, contains multiple polyketide gene clusters. , 2002, Microbiology.

[55]  H. Jenke-Kodama,et al.  Exploiting the mosaic structure of trans-acyltransferase polyketide synthases for natural product discovery and pathway dissection , 2008, Nature Biotechnology.

[56]  B. Shen,et al.  Biosynthesis of hybrid peptide-polyketide natural products. , 2001, Current opinion in drug discovery & development.

[57]  I. Furusawa,et al.  Structural analysis of PKS1, a polyketide synthase gene involved in melanin biosynthesis in Colletotrichum lagenarium , 1995, Molecular and General Genetics MGG.

[58]  B. Neilan,et al.  Evolutionary Affiliations Within the Superfamily of Ketosynthases Reflect Complex Pathway Associations , 2003, Journal of Molecular Evolution.

[59]  Kira J Weissman,et al.  The structure of docking domains in modular polyketide synthases. , 2003, Chemistry & biology.

[60]  P. Lu,et al.  Efficient disruption of a polyketide synthase gene (pks1) required for melanin synthesis through Agrobacterium-mediated transformation of Glarea lozoyensis , 2003, Molecular Genetics and Genomics.

[61]  L. Bai,et al.  The biosynthetic gene cluster of the maytansinoid antitumor agent ansamitocin from Actinosynnema pretiosum , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[62]  Gitanjali Yadav,et al.  SEARCHPKS: a program for detection and analysis of polyketide synthase domains , 2003, Nucleic Acids Res..

[63]  B. Shen,et al.  The biosynthetic gene cluster for the anticancer drug bleomycin from Streptomyces verticillus ATCC15003 as a model for hybrid peptide–polyketide natural product biosynthesis , 2001, Journal of Industrial Microbiology and Biotechnology.

[64]  H. Wolfson,et al.  Protein-Protein Interactions: Coupling of Structurally Conserved Residues and of Hot Spots across Interfaces. Implications for Docking , 2004 .

[65]  Burkhard Rost,et al.  The PredictProtein server , 2003, Nucleic Acids Res..

[66]  J. H. Yu,et al.  Sterigmatocystin biosynthesis in Aspergillus nidulans requires a novel type I polyketide synthase , 1995, Journal of bacteriology.

[67]  K. Hiratsu,et al.  The large linear plasmid pSLA2‐L of Streptomyces rochei has an unusually condensed gene organization for secondary metabolism , 2003, Molecular microbiology.

[68]  Andreas Schirmer,et al.  Cloning and characterization of a gene cluster for geldanamycin production in Streptomyces hygroscopicus NRRL 3602. , 2003, FEMS microbiology letters.

[69]  T. Stachelhaus,et al.  The specificity-conferring code of adenylation domains in nonribosomal peptide synthetases. , 1999, Chemistry & biology.

[70]  Yoram Burak,et al.  The Origins of Specificity in Polyketide Synthase Protein Interactions , 2007, PLoS Comput. Biol..

[71]  T. Leonard,et al.  Culture Conditions Control Expression of the Genes for Aflatoxin and Sterigmatocystin Biosynthesis in Aspergillus parasiticus and A. nidulans , 1998, Applied and Environmental Microbiology.

[72]  R. Haselkorn,et al.  Genes Coding for Hepatotoxic Heptapeptides (Microcystins) in the Cyanobacterium Anabaena Strain 90 , 2004, Applied and Environmental Microbiology.

[73]  Wen Liu,et al.  Biosynthesis of the Enediyne Antitumor Antibiotic C-1027 , 2002, Science.

[74]  Chaitan Khosla,et al.  Quantitative analysis of the relative contributions of donor acyl carrier proteins, acceptor ketosynthases, and linker regions to intermodular transfer of intermediates in hybrid polyketide synthases. , 2002, Biochemistry.

[75]  Rolf Müller,et al.  Formation of novel secondary metabolites by bacterial multimodular assembly lines: deviations from textbook biosynthetic logic. , 2005, Current opinion in chemical biology.

[76]  Kira J Weissman,et al.  The Structural Basis for Docking in Modular Polyketide Biosynthesis , 2006, Chembiochem : a European journal of chemical biology.

[77]  R. Birch,et al.  A multifunctional polyketide-peptide synthetase essential for albicidin biosynthesis in Xanthomonas albilineans. , 2001, Microbiology.

[78]  R. Proctor,et al.  The polyketide synthase gene pks4 from Gibberella fujikuroi encodes a key enzyme in the biosynthesis of the red pigment bikaverin. , 2002, Fungal genetics and biology : FG & B.

[79]  Kira J. Weissman,et al.  Protein—Protein Interactions in Multienzyme Megasynthetases , 2008 .

[80]  D. Nietlispach,et al.  Multienzyme docking in hybrid megasynthetases. , 2007, Nature chemical biology.

[81]  A. Trefzer,et al.  Biosynthetic Gene Cluster of Simocyclinone, a Natural Multihybrid Antibiotic , 2002, Antimicrobial Agents and Chemotherapy.

[82]  D. Cane,et al.  Dissecting and exploiting intermodular communication in polyketide synthases. , 1999, Science.

[83]  M. Daboussi,et al.  Novel Polyketide Synthase from Nectria haematococca , 2004, Applied and Environmental Microbiology.

[84]  Brian O. Bachmann,et al.  A genomics-guided approach for discovering and expressing cryptic metabolic pathways , 2003, Nature Biotechnology.

[85]  R. Proctor,et al.  A polyketide synthase gene required for biosynthesis of fumonisin mycotoxins in Gibberella fujikuroi mating population A. , 1999, Fungal genetics and biology : FG & B.

[86]  C. Walsh,et al.  Yersiniabactin synthetase: a four-protein assembly line producing the nonribosomal peptide/polyketide hybrid siderophore of Yersinia pestis. , 2002, Chemistry and Biology.

[87]  Xing-Cong Li,et al.  Production of dihydroisocoumarins in Fusarium verticillioides by swapping ketosynthase domain of the fungal iterative polyketide synthase Fum1p with that of lovastatin diketide synthase. , 2007, Journal of the American Chemical Society.

[88]  Kira J Weissman,et al.  Single Amino Acid Substitutions Alter the Efficiency of Docking in Modular Polyketide Biosynthesis , 2006, Chembiochem : a European journal of chemical biology.